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Approved methods for testing of meat & meat products of 7

Export Standards Branch | Exports Division

Approved methods for microbiological testing of meat and

meat products

(Amended August 2017)

The following is a list of Department of Agriculture and Water Resources approved test methods for

meat and meat products. From 31 January 2006 all testing of product relating to export

certification, including carcass testing under National Carcase Microbiology Monitoring Program

(formerly known as ESAM), must be by one of the methods listed with the modifications and

options specified; no other modifications are permitted. Laboratory manuals and protocols must

reflect the above and will be subject to audits to ensure compliance.

Aerobic Plate Count/Total Viable Count (TVC) Escherichia coli O157:H7 Shiga-toxin producing Escherichia coli (STEC) Generic Escherichia coli Listeria monocytogenes Salmonella

Aerobic Plate Count/Total Viable Count (TVC)

AS 5013.5-2016 Microbiology of the food chain – Horizontal method for the enumeration of microorganisms – Colony count at 30°C by the pour plate technique

AOAC 990.12 TVC Petrifilm™

AOAC 2008.10 TEMPO TVC Method: Automated Enumeration of Total Viable Count in Food

AOAC 010404 Compact Dry TC

Other methods

Any method that has been validated by an internationally recognised certification body using ISO 16140 (or equivalent i.e. AOAC guidelines) for the enumeration of total viable count in meat and meat products

Note where specific market access requirements exist for the methodology used to determine total viable count these requirements must be met

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Approved methods for testing of meat & meat products Issue: August 2017 of 7


Department of Agriculture and Water Resources

Escherichia coli O157:H7

ISO 16654:2001 Microbiology of food and animal feeding stuffs – Horizontal method for the detection of Escherichia coli O157

Note - when analysing frozen or chilled samples, the temperature of broth and samples must be at 41.5 ± 1C for a minimum of 6 h and subsequently for a further 12 to 18 hours.

FSIS MLG 5 Detection, isolation, and identification of Escherichia coli O157:H7 from meat products

Note the method has been updated changing the initial enrichment to mTSB and the definition of E. coli O157:H7. When analysing frozen or chilled samples, the temperature of broth and samples must be at 42 ± 1C for a minimum of 15 hours.

FDA BAM Chapter 4A(K)

Diarrheagenic Escherichia coli - Enrichment and isolation of E. coli Serotype O157:H7 from Foods

With the following modification; must use the IMS option and a sample size of 325 g for ground beef, analysed as five separate 65 g portions

Rapid methods

Where positive confirmation is required such confirmation must be by ISO 16654:2001, FDA BAM 4A(K) or FSIS MLG 5

Note all modifications/notes listed for each method must be followed

FSIS MLG 5A FSIS procedure for the use of Escherichia coli O157:H7 screening tests

(15-22h PCR based screening test using DuPont BAX MP)

Note the method has been updated and only mTSB should be used for enrichment of samples and the definition of E. coli O157:H7 has been changed. Note - temperature of broth and samples must be at 42 ±1C for a minimum of 15 hours.

AOAC 031002 DuPont Qualicon BAX® System PCR Assay for Real-Time E. coli O157:H7

This method is approved for 375 g composite samples in 1.5 L of BAX® System E. coli O157:H7 MP medium and incubation for 10-24 h at 42°C. Note – temperature of broth and sample must be at 42C for a minimum of 10 hours

AOAC 100701 IEH E. coli Test System for detection of non-O157 Shiga-toxin producing E. coli and E. coli O157 in raw ground beef.

Samples (375 g) are diluted in 750 mL of pre-warmed IEH enrichment medium. Incubation at 42 ± 1°C for 9-48 hours. Note ¡V temperature of broth and samples must be 42°C for a minimum of 9 hours

AOAC 2005.04 Assurance GDS for Escherichia coli O157:H7 in Selected Foods and Assurance GDS E. coli O157:H7 Tq

This method is approved for using 375 g composite samples in 1.2 L mEHEC medium and incubation for 8-18 h at 42°C. Note – temperature of broth and samples must be at 42C for a minimum of 8 hours.

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AOAC 071202 3MTM Molecular Detection Assay (MDA) E. coli O157 Method

This method is approved for 375 g composite samples in 1.5 L of BPW ISO enrichment broth and incubation for 10-18 h at 41.5 ± 1°C. Note – temperature of broth and sample must be at 41.5 ± 1°C for a minimum of 10 hours

AOAC 071001 MicroSEQ(R) Real-Time PCR System for Detection of E. coli O157:H7 in raw ground beef and beef trim

This method is approved for 375 g composite samples in 1.5 L of BPW and incubation for 16 h at 42°C. Note – temperature of broth and sample must be at 42°C for a minimum of 16 hours

The following rapid methods are not to be used for the routine testing of export meat and

meat products for E. coli O157. They are approved as backup methods for use when PCR

methods are temporarily unavailable. They may be used for the testing of product under

commercial arrangements when a methodology is not specified under that arrangement:

AOAC 996.09 BioControl VIP (8-12 h and 18-28 h options)

With the following modification: disregard plating steps for confirmation, confirmation must be by ISO 16654:2001, FSIS MLG 5.05 or FDA BAM using the IMS option. 8 h enrichment is carried out in mEHEC media

Note 18-28h option for 375g samples incubated in 1L of mTSB+n media has been validated and is approved, 8-12h option for 375g in 1L of mEHEC media has been validated and approved. Temperature of broth and samples must be at 36 ±1C for a minimum of 18 hours (for 18-28 h protocol) or at 42C for a minimum of 8 hours (8-12 h protocol).

AOAC 2000.13 Reveal (8-hours)

With the following modification: disregard plating steps for confirmation, confirmation must be by ISO 16654:2001, FSIS MLG 5.04 or FDA BAM using the IMS option.

Note longer incubation times (12-14h) are required for large samples (375g) diluted less than 1:10 in initial enrichment media i.e. in one litre. Temperature of broth and samples must be at 42C for a minimum of 8 hours.

AOAC 2000.14 Reveal (20-hours)

With the following modification: disregard plating steps for confirmation, confirmation must be by ISO 16654:2001, FSIS MLG 5.04 or FDA BAM using the IMS option.

AOAC 070201 Rapid√ for Escherichia coli O157 Lateral Flow Assay

With the following modifications, mTSB+n must be used for selective enrichment and enrichment can only be for 15 to 22h at 42±1ºC (as specified in MLG 5.05). The 8 hour enrichment option is not approved. Note – temperature of broth and samples must be at 42 ± 1C for a minimum of 15 hours.

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Shiga-toxin producing E. coli (STEC)

FSIS MLG 5B Detection and isolation of non-O157 Shiga-toxin Producing Escherichia coli (STEC) from meat products

Rapid methods

Where positive confirmation is required such confirmation must be by FSIS MLG 5B

AOAC 071301 Assurance GDS® MPX Top 7 STEC for detection of top 7 pathogenic STEC in beef trim

Samples (375 g) are diluted in 1.5 L of pre-warmed (42C) mEHEC medium. Incubation is carried out for 10 h at 42C. Note – temperature of broth and samples must be 42 ± 1C for a minimum of 10 hours

AOAC 091301 DuPont Qualicon BAX® System Real-Time PCR Assays for detection of selected STEC in beef trim

Samples (375 g) are diluted in 1.5 L pre-warmed (45-46C) Bax® System MP enrichment broth. Samples are incubated at 39-42C for 12-24 h. Note – temperature of broth and samples must be at 39-42C for a minimum of 12 hours.

AOAC 0100701 IEH E. coli Test System for detection of non-O157 Shiga-toxin producing E. coli and E. coli O157 in raw ground beef

Samples (375 g) are diluted in 750 mL of pre-warmed IEH enrichment medium. Incubation at 42 ± 1C for 9-48 hours. Note – temperature of broth and samples must be 42C for a minimum of 9 hours

AOAC 061602 RapidFinderTM STEC Detection Workflow for detection of top 7 STEC serogroups in beef products.

Samples (375 g) are diluted in 1.0 L of pre-warmed (48C) Trypticase Soy Broth. Incubation at 42C for 8 hours. Note – temperature of broth and samples must be at 42C for a minimum of 8 hours

AOAC 031401 Pall GeneDisc(R) Plate STEC Top 6 methods for detection of O157 and top 6 non-O157 Shiga toxin producing E. coli in raw ground beef and beef trim

Samples (375 g) are diluted in 1.5 L of pre-warmed (41.5 ± 1C) BPW Broth. Incubation at 41.5 ± 1C for 10-20 hours. Note – temperature of broth and samples must be at 41.5 ± 1C for a minimum of 10 hours

AOAC 101502 Assurance GDS® MPX ID for Top 6 STEC

Detection of Top 6 Shiga toxin-producing E. coli (O26, O45, O103, O111, O121 and O145) in beef trim as a secondary screening method following a positive result using the Assurance GDS® MPX Top 7 STEC assay (AOAC 071301). All screen positive samples must be confirmed by MLG 5B.

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Generic Escherichia coli

AS 5013.15 General guidance for enumeration of presumptive Escherichia coli - Most probable number technique

Note this is an update from the 2004 standard, the main change being that the temperature of incubation is now 44±1ºC

AOAC 991.14 E. coli PetriFilm™

Butterfields or buffered peptone water or 0.1% Peptone Salt Solution must be used as diluent

AOAC 998.08 E. coli PetriFilm™

Butterfields or buffered peptone water or 0.1% Peptone Salt Solution must be used as diluent

AOAC 2005.03 SimPlate® Colour Indicator: Detection and Quantitation of Coliforms and E. coli in foods

AOAC 2009.02 Tempo® EC AFNOR Bio 12/13 – 02/05 for testing of generic E. coli

AOAC 110402 Compact Dry EC

Listeria monocytogenes

AS 5013.24.1 Food and animal feeding stuffs – Horizontal method for the detection and enumeration of Listeria monocytogenes. Detection method

FSIS MLG 8 Isolation and identification of Listeria monocytogenes from red meat, poultry, egg, and environmental samples

Note alternative secondary enrichment has been included

Rapid methods

Where positive confirmation is required such confirmation must be by Australian Standard AS 5013.24.1 or FSIS MLG 8

Note the following bio-chemical test systems can be used for confirmation for all methods MICRO-ID® Listeria or API®-Listeria or VITE® 2 Compact, . β-lysin. CAMP factor discs (Remel #21-120, or equivalent) can be used instead of the traditional CAMP test procedure

FDA BAM Ch 10 Detection and Enumeration of Listeria monocytogenes in Foods

FSIS MLG 8A FSIS procedure for the use of Listeria monocytogenes BAX screening test

Note the method has been updated to include testing of liquid egg products

AOAC 2003.12 Automated BAX System for Detection of Listeria monocytogenes in Foods

AOAC 999.06 & AOAC 2004.06

VIDAS LIS Assay for Listeria in Food

AOAC 995.22 TECRA Listeria Visual Immunoassay

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AOAC 2002.09 TECRA Listeria Visual Immunoassay

AOAC 997.03 BioControl Listeria Visual Immunoprepicipate (VIP) Assay

AOAC 996.14 BioControl Assurance Listeria Immunoassay

AOAC 070401 Foodproof Listeria moncocytogenes Detection Kit, 5’Nuclease and Hybridization Probes

AOAC 031204 Pall GeneDisc(R) method for the detection of Listeria monocytogenes in food and environmental samples

AOAC 081203 3MTM Molecular Detection Assay (MDA)

AOAC 011022 MicroSEQ(R) Real-Time PCR System for Detection of Listeria monocytogenes in food

NF BIO 12/33-05/12

VIDAS UP Listeria method (VIDAS LPT)

NF SOL 37/02-06/13

Solus Listeria ELISA

AOAC 071304 Thermo ScientificTM SureTectTM Listeria species PCR Assay - AOAC 071304 (AFNOR UNI 03/09 - 11/13)

AOAC 121402 DuPontTM BAX® System Real-Time PCR Assay for Listeria monocytogenes - AOAC 121402

Salmonella

AS 5013.10 Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp

The following options are required: Second agar choice must be capable of detecting H

2S negative Salmonella (internationally validated Salmonella media

e.g. BGA, BGS, Rambach, ChromAgar)

FSIS MLG 4 Isolation and identification of Salmonella from meat, poultry and egg products

Rapid methods

When positive confirmation is required such confirmation must be by Australian Standard AS 5013.10-2009 or FSIS MLG 4.04

FSIS MLG 4C FSIS procedure for the use of the BAX system PCR assay for screening Salmonella in raw meat, carcass sponge samples, whole bird rinses, ready-to-eat meat and poultry products and pasteurised egg products

Note procedure to follow when a PCR indeterminate or signal-error occurs has been updated

AOAC 2003.09 BAX Automated System for Screening Salmonella in foods




AOAC 992.11 BioControl Assurance EIA

With instructions specified for pre-enrichment in table 999.08 C (ie use of BPW + novobiocin)

AOAC 999.08 BioControl Assurance Gold

pre-enrichment with BPW + novobiocin as per instructions

AOAC 999.09 BioControl VIP

With instructions specified for pre-enrichment in table 999.08 C (ie use of BPW + novobiocin)

AOAC 989.14 & AOAC 998.09

TECRA Visual Immunoassay

With the following modification: pre-enrichment in buffered peptone water (BPW)

AOAC 998.09 TECRA ULTIMA

With the following modification: pre-enrichment in BPW

AOAC 996.08 VIDAS Salmonella (SLM) Assay

AFNOR BIO 12/16–09/05

VIDAS EASY Salmonella method – AFNOR

AOAC 071101 VIDAS UP Salmonella method (VIDAS SPT)

AOAC 2001.09 VIDAS Immuno Concentration Salmonella (ICS)

AOAC 2009.03 Assurance GDS™ Salmonella method for foods and Assurance GDS Salmonella Tq method

AOAC 100701 IEH PCR assay for detection of Salmonella in carcass and environmental sponges or swabs

AOAC 120301 foodproof Salmonella Detection Kit, 5’Nuclease and Hybridization Probes

AOAC 100201 DuPont Qualicon BAX (R) System Salmonella2 PcR Assay

AOAC 031208 3MTM Molecular Detection Assay (MDA) Salmonella Method

AOAC 2013.02 DuPont Qualicon BAX(R) System real-time PCR assay for Salmonella

AOAC 031001 MicroSEQ(R) Real-Time PCR System for Detection of Salmonella in food

AOAC 050602 Assurance GDS for Salmonella

AOAC 2014.01 3M Petrifilm Salmonella Express

AOAC 011404 VeriflowTM Salmonella Species (SS)

NF SOL 37/01-06/13

Solus Salmonella ELISA

AOAC 051303 Thermo ScientificTM SureTectTM Salmonella spp PCR Assay - AOAC 051303 (AFNOR UNI 03/07 – 11/13)

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